Abstract Head and Neck Squamous Cell Carcinoma (HNSCC) is the sixth leading incidence of cancer worldwide. Standard therapies can be effective for site-specific subsets, but overall the 5-year survival rate is 40-50%. We hypothesize that significant improvements in patient survival will require the development of precision medicine protocols, in which treatments are designed to target the specific disease process of the individual patient. The potential for targeted therapy in HNSCC has been shown with the Epidermal Growth Factor Receptor (EGFR) inhibitor Cetuximab, in which Cetuximab and radiation extended patient survival by 19.3 months in comparison to radiation alone. However, as the sensitivity and resistance mechanisms are unknown, there are no biomarkers that predict response to targeted EGFR inhibition. Likewise, patients that relapse from this therapy often have highly aggressive and rapidly lethal recurrences. A better understanding of the compensatory pathways that rescue tumor growth is needed to devise more effective combination strategies and improve patient survival. Our central hypothesis is that there are specific compensatory pathways containing targetable genes that drive resistance to EGFR inhibition, and that disruption of these genes will overcome resistance to EGFR-targeted therapies. Our initial study used the Genome CRISPR Knock-Out (GeCKO) library, a CRISPR/Cas9 library that created a stable pool of 18,000+ genetic knock-outs, and we observed that knock-outs in the Fibroblast Growth Factor Receptor (FGFR) pathway sensitized cells to a low dosage of an EGFR inhibitor in a subset of models. Further preliminary data indicates that 40% of our HNSCC models that are resistant to EGFR inhibition are sensitive to the combination of EGFR and FGFR inhibition. For this proposal, we will extend this observation to assess the mechanistic role of FGFR kinase compensation in rescuing cell survival during EGFR inhibition, as well as identify novel mechanisms of resistance to EGFR inhibitors in the remaining 60% of models. In identifying and understanding the compensatory pathways that drive resistance to EGFR inhibition, this proposal will help to develop precision medicine protocols using combination therapies for patients with HNSCC.